Abstract

Abstract Understanding the genetic control of cardiac development has been greatly assisted by the newly acquired ability to generate targeted mutations in the mouse. A number of mutations in genes that directly or indirectly affect cardiac development have now been reported, and the phenotypes of these mutations have suggested that cardiac development is under complex genetic control. Further analysis of the mouse model system should help elucidate the etiology of human congenital cardiovascular anomalies.

Congenital cardiovascular anomalies are the most common form of human birth defect, with a recorded instance of 1 per 200 live births each year in North America. Since a functioning circulatory system is a prerequisite for survival of the fetus to term, many cases of preterm loss must also be ascribed to cardiovascular defects. However, there is still little information on the etiology of most human cardiovascular anomalies.

The mouse provides a useful model system for studying the development of the cardiovascular system because of the detailed embryological information available and the ability to genetically manipulate its genome. Accumulated information has pinpointed several critical time points during mouse gestation when embryonic death can occur.1 Most of these are related to critical periods in the development of the cardiovascular system, whereas gross anomalies in the development of other organ systems such as the central nervous system or the lungs are compatible with survival to term. There are several aspects of the development of the cardiovascular system that are susceptible to disruption leading to embryonic mortality. During the early stages of postimplantation development, the embryo obtains its nutrition from the uterus by passive diffusion. However, as the embryo grows, passive diffusion is not enough, and the development of the yolk sac circulation is essential for transport of nutrients absorbed by the yolk sac to the developing fetus. Formation of the yolk sac blood islands, which will form both hematopoietic progenitors and endothelial precursors, and development of the heart itself, which will drive the circulation, are thus critical for early survival of the fetus. Once the embryo has reached a certain size (around E9.5 in the mouse), the yolk sac circulation also becomes limiting, and the establishment of a more direct link between the fetal and maternal circulation via the placenta is required. In the mouse, this occurs by fusion between the allantois and the chorion to form the chorioallantoic placenta. Failure of this fusion leads to embryonic mortality around E10.5. Once the embryo has passed these critical points, there are still many other circulatory problems that can lead to embryonic morbidity and mortality. Inefficient pumping action by the heart may lead to circulatory failure at various stages. Instability of the blood vessel walls may cause hemorrhage and death. Inappropriate remodeling of the major blood vessels can also cause problems, especially at birth, when the pulmonary circulation must be established.

In the mouse, there has been a rapid recent accumulation of new single-gene mutations generated by targeted mutagenesis using homologous recombination in embryonic stem cells.234 Because of the sensitivity of the cardiovascular system to disruption leading to lethality, a considerable number of these new mutations have been reported to show defects in the development of the heart or the vascular system. They thus provide a rich source of new information concerning the genetic regulation of cardiovascular development as well as the possible etiology of human cardiovascular anomalies. In this review, I will focus only on those genes whose disruption by targeted mutagenesis has been reported to cause defective development of the heart itself and not on those whose primary role seems to be in the development of the vascular system or the placenta.

It is hard to discern the detailed genetic strategies of heart development from all the disparate information available at this time. However, some commonality of phenotypic effects has been observed, pointing to critical steps in cardiogenesis. The heart is the first organ to develop and function in the fetus, a process requiring the coordinated development of cells derived from several different embryonic lineages, including the myocytes of the myocardium, the endothelial cells of the endocardium, and the cells of the neural crest that form the outflow tracts. Several mutations appear to directly affect the development of the myocardium and may identify critical regulators of the lineage. Mutational analysis has also identified several genes whose roles seem to be primarily in the neural crest derivatives. Further, mutation of genes involved in cell adhesion and in cell-cell communication has shown that the coordination of intercellular events is critical for cardiac development. Beyond these examples (described in more detail below), in which a primary effect on heart development can be fairly clearly demonstrated, there are an ever-increasing number of mutations in which cardiac failure is cited as the cause of death but in which the etiology of the defect is unclear at this time. A list of mutations in which cardiac anomalies have been reported is provided in the Table⇓.

Transcription Factors in the Myocardium

There is considerable interest in understanding the genetic regulation of the myogenic lineage of the heart, which is specified soon after gastrulation in the anterior mesoderm region of the embryo. Despite the fact that mammalian cardiac and skeletal muscle cells express several myofilament proteins in common, the molecular mechanisms that have been so elegantly defined for the skeletal myogenic lineages do not appear to apply to the cardiac lineage.27 The myogenic regulatory factors of the myoD-related basic helix-loop-helix family are not expressed in heart muscle, and mutations in these genes do not affect cardiac development.28293031 Presumably, other regulatory pathways are used. Multiple transcription factors have been implicated in cardiac myocyte–specific gene regulation in vitro, but so far, only a few of these factors have proved to be specific to the cardiogenic mesoderm and are expressed early enough to be prime candidates as master regulators of the cardiac muscle lineage. GATA4,32Mef2C,33 and Nkx2-53435 are all expressed in cardiogenic progenitors, and recent mutational analysis supports a role for Nkx2-5, at least, in normal heart morphogenesis.

Nkx2-5 is a member of the NK class of homeodomain proteins and is homologous to the tinman gene,36 which is involved in heart development in Drosophila. Embryos homozygous for a disruption of the Nkx2-5 gene initiate heart formation but fail to undergo normal looping morphogenesis at E8.5.12 Embryos die soon after, apparently from circulatory insufficiency. Since cardiac myocytes clearly develop in the mutant embryos, Nkx2-5 is not required for cells to enter the cardiac myogenic lineage, but it is required for full development of the myocardial phenotype, especially in the ventricular region. Although mutant cardiomyocytes do express a number of specific markers, they fail to express the ventricular-specific myofilament gene MLC2V. This implicates Nkx2-5 in cardiac myogenic gene regulation but also indicates that it is not a “master regulator,” coordinately regulating sets of cardiac-specific genes. However, since there are multiple Nkx-like genes in mammals, which may have some overlapping functions with Nkx2-5,36 a predominant role for this gene family in cardiac muscle specification cannot yet be excluded.

Another gene that has been shown to be involved in cardiac myocyte gene regulation by in vitro studies is the broadly expressed transcription factor TEF-1. A mutation in TEF-1 generated by insertion of a retroviral gene trap vector, not by homologous recombination, does appear to affect normal myocardial development.20 Homozygous embryos die between E11 and E12 and show an abnormally thinning of both the compact layer and the trabeculae of the ventricular wall, leading to cardiac insufficiency. Since TEF-1 has been shown to regulate some cardiac-specific genes, such as cardiac troponin C, T, and I, as well as the myosin heavy chain genes,37 at first glance this phenotype would seem to implicate TEF-1 as a critical regulator of cardiac gene expression. However, the genes known to be regulated by TEF-1 in vitro are still expressed at relatively normal levels in the mutant hearts. Thus, the exact role of TEF-1 in cardiac development is still unclear, nor is it apparent why the sole defect observed in these embryos is in the heart, given the wide expression and proposed functions of the TEF-1 protein.

Mutations in two transcription factor genes, for which there was no prior indication of a possible role in myocardial gene regulation, also cause defects that appear to primarily affect the development of the myocardial layers of the heart. Several groups reported that a null mutation in the N-myc gene causes embryonic lethality around E11 with multiple developmental defects,131415 including poor development of the ventricular myocardium and the interventricular septum. Compound heterozygotes carrying both a null and a hypomorphic allele at the N-myc locus survived a little longer than the null mutants and showed a clear cardiac defect,16 in which the outer compact layer of the ventricular myocardium, but not the inner trabeculae, was deficient. This defect apparently caused cardiac pump failure and fetal death. N-myc is expressed specifically in the compact layer of the myocardium, suggesting that reduction in the myocardium is a primary response to the loss of N-myc. Although N-myc, like other myc genes, is known to be able to trans-activate genes by binding to specific DNA consensus sequences as a heterodimer with the protein, Max,3839 its downstream targets are not known, and so its precise role in the genetic regulation of the myocardium remains to be elucidated.

A very similar cardiac phenotype was observed in embryos carrying a null mutation in RXR-α.1819 Retinoid receptors are members of the steroid hormone receptor–related family of ligand-dependent zinc-finger transcription factors.4041 The RXRs can form cooperative heterodimers with RARs, as well as other members of the hormone receptor superfamily,4243 and are thus thought to play a pivotal role in mediating the effects of several hormonal signals. Despite the evidence for an involvement of retinoic acid in various aspects of development, mutating individual RARs did not result in serious developmental defects.4445464748 Mice mutant for RXR-α, on the other hand, died around E14 from cardiac failure.1819 The compact layer of the myocardium and the ventricular septum were drastically thinned, as seen in N-myc mutants. Embryonic vitamin A deficiency causes a similar defect,49 supporting the idea that it is specifically the retinoid signaling pathway that is disrupted in the RXR-α mutants. Additional defects were seen in various compound mutants between RXR-α and the RARs.19 These defects included persistent truncus arteriosus and aortic arch defects, suggestive of neural crest disruption. Genetic evidence thus supports an involvement of retinoic acid signaling in the development of both the myocardium and the neural crest cells of the developing heart.

Thus, mutations in three rather widely used transcription factors, as well as one cardiac-specific factor, have indicated critical roles for these genes in the development of the myocardium. How these genes fit into the hierarchy of gene control of cardiac myocyte development remains to be seen. Unlike the situation with the development of skeletal muscle, there are relatively few cardiac-specific transcription factors identified so far, and it may be that interactions between specific factors and more general factors, like N-myc and TEF-1, play a more predominant role in the heart than in skeletal muscle.

Neural Crest Defects

A number of mouse mutations have been reported to be associated with the kinds of cardiovascular anomalies that suggest defects in the development of the neural crest component of the heart. For example, mutations in two transcription factor genes, Hoxa3 and Pax3, cause pleiotropic effects, including heart anomalies. Hoxa3 is a member of the Hox gene families, which are involved in establishing positional identity along the body axis.50 Mice that are homozygous for a mutation in the Hoxa3 gene survive to term but show multiple anomalies, including defects in the atria, heart valves, and major vessels.8 There is considerable evidence that patterning of the segmentally arrayed hindbrain rhombomeres and their associated neural crest is regulated by the Hox genes50 ; thus, defects in the neural crest–derived heart structures are not unexpected. It is important to note that a single gene mutation in a critical patterning gene can produce such a wide spectrum of developmental anomalies, since it suggests that complex congenital anomaly syndromes in humans may have simple underlying causes.

In humans, a single gene defect underlies the multiple anomalies in neural crest–derived tissues, including the heart, that typify Waardenburg’s syndrome. These patients are heterozygous for a mutation in Pax3,51 a transcription factor gene related to the Drosophila paired gene. Interestingly, mice carrying mutations in this gene also exist,52 not produced by targeted mutagenesis but arising as spontaneous mutations—the Splotch mutants. Among the neural crest phenotypes seen in homozygous Splotch mutants is persistent truncus arteriosus,20 and homozygous embryos die around E13.5 with symptoms of congestive heart failure. Pax3 is strongly expressed in the developing head neural crest as well as in other tissues53 and seems to play a critical role in directing neural crest development.

Humans heterozygous for mutations in the NF1 tumor suppressor gene exhibit the disease neurofibromatosis, in which benign and malignant abnormalities occur in a variety of neural crest–derived tissues.54 This suggests that NF1 could also be an important player in neural crest development. The NF1 gene encodes a protein, neurofibromin, that acts as a negative regulator of the Ras protein in signal transduction,5556 and is widely expressed during development.57 Mouse embryos homozygous for a mutation in the NF1 gene die around day 14 of development and show multiple defects in organ development as well as hyperplasia of the sympathetic ganglia.1011 Death is attributed to cardiac failure, as evidenced by generalized edema and vascular congestion. The heart is hypoplastic, and the muscle cells are disorganized. Septal defects, persistent truncus arteriosus, and valve abnormalities were also observed. These latter defects are typical of neural crest defects. The associated defects in neural crest–derived ganglia support the idea that the NF1 mutation may have a primary effect in the neural crest cell lineage. Since Ras signaling has been implicated in many developmental processes, it will be interesting to elucidate which signal transduction pathways are regulated by NF1 in the neural crest lineage and how such pathways intersect with nuclear factors like Pax3 and the Hox genes.

Cell-Cell Adhesion and Cell-Cell Communication

Production of a complex organ like the heart requires cells to communicate and coordinate their activity in order to maintain structural and functional integrity. Interactions between extracellular matrix molecules and their receptors, the integrins, have been proposed to play many roles in tissue and organ interactions. Mutations in two such genes, the cell adhesion molecule, VCAM-1,2425 and a component of its receptor, α4-integrin,5 have been shown to cause specific defects in cardiovascular development. In both ligand and receptor mutants, some embryos die around E10.5 because of failure of fusion of the chorion and allantois and subsequent failure to develop the chorioallantoic circulation. α4-Integrin is expressed on the chorion, whereas VCAM-1 is expressed on the allantois, suggesting that direct interaction between these molecules is a normal part of the process of chorioallantoic fusion. Some embryos manage to circumvent this defect and achieve fusion by other pathways. These embryos mostly die around E12 of cardiac and perhaps placental problems. The most obvious defect in the heart is the breakdown of the epicardium, the thin layer of mesothelial cells overlying the compact layer of the myocardium. Coronary vessels normally develop in the subepicardial space; such vessels are missing in the mutant hearts, and blood is observed in the pericardial cavity. α4-Integrins are expressed by the epicardial layer, and VCAM-1 is expressed by the underlying myocardium, suggesting that a direct interaction between these layers is required to promote the integrity of the epicardium and to allow coronary vessels to develop. Whether there are also effects on the myocardium itself is less clear. One group reports thinning of the myocardium in VCAM-1 mutants,25 but this could simply result from overall developmental delay caused by circulatory problems.

Another form of cell-cell communication thought to be critical for the development of many organs, especially the heart, is gap junctional communication. Gap junctions are channels between cells made up of hexamers of proteins called connexins, which allow the passage of ions and small molecules between cells. There are at least 12 different connexin proteins expressed in various regions of the body.58 In the heart, the connexin43 gene is strongly expressed in cardiac muscle5960 and has been postulated to be critical for the conductance properties of cardiac muscle that maintain the coordinated contraction of the heart. Surprisingly, embryos homozygous for a mutation in the connexin43 gene survive to term and show apparently normal contraction of the heart.6 Other connexins, including connexin40 and connexin45, are also expressed in the heart, and it will be necessary to determine whether these connexins can compensate for the loss of connexin43. Connexin43 mice do, however, show a cardiac defect involving a swelling and blockage of the right ventricular outflow tract. This defect causes perinatal mortality that is due to an inability to establish the pulmonary circulation. Exactly why the loss of connexin43 should produce this very specific defect is still unclear. It could still be an indirect consequence of some change of conductance in the cardiac muscle that causes abnormal mechanical load and hence abnormal morphogenesis. However, since the outflow tracts are largely populated by neural crest cells, which undergo complex remodeling during development, gap junctional communication could be involved in coordinating this process.

Future Directions

This brief summary should serve to illustrate the wealth of new material relevant to the heart that is produced by the analysis of mouse mutants. Several mutants have pointed to the direct involvement of specific genes in specific aspects of cardiac development, whereas other mutations have suggested that the normal function of the heart may depend on factors acting outside of the heart tissue itself. The mutations described in the present review are almost all severe lack-of-function mutations, and the phenotypes are concomitantly severe, often resulting in preterm lethality. Milder mutations in the same genes may cause less drastic alterations in cardiac function that may be more similar to the human congenital anomalies observed in infants at term and beyond. Technologies exist to make such mutations in the mouse, and it will be interesting to compare and contrast effects seen in the mouse with some of the human syndromes. Future genetic analysis of mouse mutations will provide a clearer picture of the primary and secondary influences that determine the normal development of the cardiovascular system.

Selected Abbreviations and Acronyms

E (associated with number)

=

embryonic day

RAR

=

retinoic acid receptor

RXR

=

retinoid X receptor

TEF

=

transcriptional enhancer factor

VCAM

=

vascular cell adhesion molecule

Acknowledgments

The author’s own work quoted here was supported by grants from the National Cancer Institute of Canada (Canadian Cancer Society and Terry Fox Foundation) and the Medical Research Council of Canada. Dr Rossant is a Terry Fox Cancer Research Scientist and a Howard Hughes International Scholar.